E2F-1基因沉默对舌鳞癌细胞环氧化酶-2基因表达影响的研究

Influence of RNA interference lentiviral vector in E2F-1 on COX-2 in Tca8113 cells

目的探讨核转录因子E2F-1基因沉默对舌鳞癌细胞环氧化酶-2(COX-2)基因表达的影响。方法体外合成靶向E2F-1基因的短发卡状RNA(short hairpin RNA,shRNA)序列,克隆到pLKO.1-PURO-U6(Age I/EcoR I)质粒载体中,经酶切和测序鉴定所构建的重组载体,将以上质粒分别和包装质粒混合物共转染293T细胞,包装产生病毒颗粒。将病毒载体转染舌鳞癌细胞Tca8113细胞后,运用Real-Time PCR和Western-blot检测E2F-1和COX-2基因的表达。结果经酶切和测序证明,靶向E2F-1基因的shRNA序列正确;转染组E2F-1和COX-2基因mRNA和蛋白表达均发生明显下调,与空白对照组相比,差异有统计学意义,阴性对照组未见明显差异。结论靶向E2F-1基因的shRNA慢病毒载体可特异性沉默E2F-1基因的表达;E2F-1基因沉默后可下调COX-2基因表达。

Objective To investigate the influence of RNA interference lentiviral vector in E2F-1 on COX-2 in Tca8113 cells.Methods DNA oligonucleotides targeting gene E2F-1 on appropriate site and one pair of negative control oligonucleotide sequence were synthesized and inserted into pLKO.1-PURO-SP6（Age I/EcoR I） vector.The sequences of two plasmids were identified by DNA sequencer and restriction endonuclease digestion.These recombinant plasmids were transfected into the 293T cells along with lentiviral packing mix by lipofectamine 2000 for the package of lentiviral particles.Then the lentiviral vector particles were transfected into Tca8113 cells and E2F-1 expression in the transfected cells was assayed with Real-Time PCR.The expression of E2F-1 and COX-2 protein was examined by Western-blot.Results It was verified that the specific DNA oligonucleotide was cloned into the vector successfully and the expression of E2F-1 mRNA in Tca8113 cells was significantly reduced after the recombinant plasmids were transfected,compared to the control group.（P0.05）.The expression of E2F-1 and COX-2 protein was significantly reduced after transfection.Conclusions E2F-1 gene silencing suppresses the expression of COX-2,indicating COX-2 is the target gene of E2F-1.